The juxtaglomerular apparatus of the kidney is a structure that connects the renal distal tubule with the glomerular vascular pole. It serves as a site of monitoring the delivery of NaCl to the late renal nephron, and it counteracts potential salt losses by reducing filtration and by controlling the release of renin. Renin is synthesized in the JGA and its release is critical for salt balance because it is rate-limiting in the formation of angiotensin II, probably the most important salt-regulating peptide. The signaling events leading to a change in the secretion of renin are initiated by the epithelial cells at the tubulo-vascular contact point, the macula densa (MD) cells. To study the function of the MD cells we established for the first time a line of these cells by labelling with an MD specific fluorescent lectin, FAC sorting, cloning of individual cells, and identification of MD characteristic markers. Using these cells we determined that a reduction of medium NaCl concentration caused a prompt release of PGE2 and a delayed increase in the expression of cyclooxygenase-2 (COX-2), and this response was dependent on the reduction of Cl rather than Na concentration. The effect was mimicked by inhibition of the Na,K,2Cl cotransporter. A reduction in medium NaCl concentration did not alter cytosolic Ca concentration, but it did cause a rapid phosphorylation of the p38 and Erk1/2 mitogen activated kinases. Inhibition of these kinases prevented the upregulation of COX-2 suggesting that the stimulation of COX-2 expression involves the MAP kinase pathway. MD-mediated stimulation of renin secretion is initiated by enhanced COX-2 activity and COX-2 expression, subsequent release of PGE2, and interaction with prostaglandin receptors on granular cells of the JGA. Inhibition of nitric oxide also inhibited enhanced PGE2 release and COX-2 expression in MD cells exposed to a low NaCl medium indicating an interaction between NO and COX-2.